This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In this project we seek to establish the relationship between the dynamics and the catalytic properties of a model enzyme (subtilisin Carslberg) dissolved in organic solvents. Our preliminary EPR data obtained with the active site TEMPO spin-labeled serine protease subtilisin Carlsberg suspended in several organic solvents show a two component spectra that changes during the time the enzyme is exposed to organic media. Furthermore, similar observations made using FRET and Fluorescence spectroscopy lead us to hypothesize that a change in the polarity of the active site environment induces substrates (and active site inhibitors such as TEMPO) to adopt a different binding orientation during prolonged exposure to organic media. We are presently using MOMD fitting program, NLSL.MOMD, to evaluate multiple EPR data obtained with our model enzyme suspended in four different organic solvents during different solvent exposure times. The two-component EPR spectra that change with exposure time are characterized by rotational diffusion rates and orienting potential of each component. We anticipate one manuscript to emerge from this project.